(i) Field of the Invention
The present invention relates to genetically modified plant cells and plants, and to processes for the production of genetically modified plant cells and plants which have an increased activity of a protein having the activity of a starch synthase II and an increased activity of a protein having the activity of a glucan-water dikinase. Plants of this type synthesize starches having increased hot water swelling power. The present invention likewise relates to starches having increased hot water swelling power, and to processes for their preparation.
(ii) Description of the Related Art
Beside oils, fats and proteins, polysaccharides are the main renewable raw materials of plants. Starch, which is one of the most important reserve substances in higher plants, beside cellulose, takes on a central role in the polysaccharides.
Furthermore, starch is an essential constituent of human and animal nutrition in nutritional physiology terms. The structural features of the starch contained in foods can influence the functional (e.g. water-binding power, swelling power), nutritional physiology (e.g. digestibility, influence of the food on the glycemic index) or structure-imparting (e.g. cut resistance, texture, stickiness, processability) properties of all sorts of foods. Food compositions therefore often contain a starch having certain structural features which determine the desired properties of the food in question. The properties of foods containing starch-storing plant tissue (e.g. grains, fruit, flours) can also be influenced by the starch contained in the plant tissues.
The polysaccharide starch is a polymer of chemically homogeneous basic structural units, the glucose molecules. What is involved here, however, is a very complex mixture of different molecular forms, which differ with respect to their degree of polymerization, the occurrence of branchings of the glucose chains and their chain lengths, and which can moreover be modified, e.g. phosphorylated. Starch is therefore not a homogeneous raw material. In particular, amylose, an essentially unbranched polymer of alpha-1,4-glycosidically linked glucose molecules, is distinguished from amylopectin, which is a complex mixture of differently branched glucose chains. The branchings come about here as a result of the occurrence of additional alpha-1,6-glycosidic linkages. In typical plants used for industrial starch production or as foods, such as, for example, corn, rice, wheat or potatoes, the synthesized starch consists to about 20%-25% of amylose and to about 70%-75% of amylopectin.
The functional, nutritional physiology or structure-imparting properties of the starch, such as, for example, the solubility, the retrogradation behavior, the water-binding capacity, the film formation properties, the viscosity, the gelatinization properties, the freeze-thaw stability, the acid stability, the gel strength, the swelling power, the digestibility and the starch granule size of starches are influenced, among other things, by the structural features of the starch such as the amylose/amylopectin ratio, the molecular weight of the glucose polymers, the pattern of side chain distribution, the content of ions, the lipid and protein content and/or the starch granule morphology etc.
By means of processes based on breeding, selected structural features of the starch and thus also functional, nutritional physiology or structure-imparting properties of starch in plant cells can be altered. However, this is only possible today for selected structural features of starch (e.g. amylopectin/amylose content, U.S. Pat. No. 5,300,145). At present, for example, it is not possible to influence the content of phosphate in plant starch alone by breeding measures.
An alternative to breeding processes consists in the selected modification of starch-producing plants by genetic engineering methods. A prerequisite for this, however, is the identification and characterization of the enzymes involved in starch synthesis and/or starch modification and their subsequent functional analysis in transgenic plants.
Various enzymes which catalyze different reactions are involved in starch synthesis in plant cells. Starch synthases (EC2.4.1.21, ADP-glucose, 1,4-alpha-D-glucan 4-alpha-D-glucosyltransferase) catalyze a polymerization reaction by transfer of a glucosyl radical of ADP-glucose to alpha-1,4-glucans, the transferred glucosyl radical being linked to the alpha-1,4-glucan by production of an alpha-1,4 bond. In almost all plants investigated up to now, it was possible in each case to demonstrate a number of isoforms of starch synthases. Starch synthases can be divided into two different groups: granule-bound starch synthases (GBSS) and soluble starch synthases (also abbreviated as “SS” in connection with the present invention). Granule-bound starch synthases catalyze the synthesis of amylose, whereas soluble starch synthases are involved in the synthesis of amylopectin (Ball and Morell, 2003, Annu. Rev, Plant Biol. 54, 207-233; Teltow et al., 2004, J. Expt. Bot. 55(406), 2131-2145). The group of soluble starch synthases has a number of isoforms which are designated in the technical literature as SSI, SSII, SSIII, SSIV. The assignment of starch synthases to the individual groups (SSI, SSII, SSIII, SSIV) is carried out by means of sequence homologies of the protein sequences of the respective enzymes in question (Ball and Morell, 2003, Annu. Rev, Plant Biol. 54, 207-233). Each individual isoform of the soluble starch synthases is assigned a specific function in starch synthesis according to current doctrine. In dicotyledonous plants, up to now it was only possible to demonstrate one isoform of SSII proteins, whereas in many monocotyledonous plants (e.g. corn) two different classes of SSII proteins were demonstrated, which are designated by SSIIa or SSIIb. In monocotyledonous plants, SSIIa is expressed preferentially in the endosperm and SSIIb preferably in the leaf tissue (Teltow et al., 2004, J. Expt. Bot. 55(406), 2131-2145). The specific function, in particular of the individual soluble starch synthases in the synthesis of starch, is at present still not finally clarified (Ball and Morell, 2003, Annu. Rev, Plant Biol. 54, 207-233).
The functional, nutritional physiology or structure-imparting properties of starch are also influenced by the phosphate content, a non-carbon component of starch. A distinction is to be made here between phosphate which is covalently bonded to the glucose molecule of the starch in the form of monoesters (in connection with the present invention designated as starch phosphate) and phosphate in the form of phospholipids associated with the starch.
The content of starch phosphate varies depending on the type of plant. For instance, certain corn mutants synthesize a starch having an increased content of starch phosphate (waxy corn 0.002% and high amylose corn 0.013%), while conventional types of corn only contain traces of starch phosphate. Likewise, small amounts of starch phosphate are found in wheat (0.001%) while in oats and Sorghum it was not possible to detect any starch phosphate. Less starch phosphate was likewise found in rice mutants (waxy rice 0.003%) than in conventional types of rice (0.013%). Significant amounts of starch phosphate were found in plants synthesizing tuber or root store starch such as, for example, tapioca (0.008%), sweet potato (0.011%), arrowroot (0.021%) or potato (0.089%). The percentage values for the starch phosphate content cited in the preceding text in each case relate to the dry weight of the starch and have been determined by Jane et al. (1996, Cereal Foods World 41 (11), 827-832).
Starch phosphate can be present in the form of monoesters in the C2, C3 or C6 position of the polymerized glucose monomers (Takeda and Hizukuri, 1971, Starch/Stärke 23, 267-272). The phosphate distribution of the phosphate in starch synthesized by plants is distinguished in general in that approximately 30% to 40% of the phosphate radicals in the C3 position and approximately 60% to 70% of the phosphate radicals in the C6 position of the glucose molecules are covalently bonded (Blennow et al., 2000, Int. J. of Biological Macromolecules 27, 211-218). Blennow et al. (2000, Carbohydrate Polymers 41, 163-174) determined a content of starch phosphate which is bonded in the C6 position of the glucose molecules for various starches, such as, for example, potato starch (between 7.8 and 33.5 nmol per mg of starch, depending on cultivar), starch from various Curcuma species (between 1.8 and 63 nmol per mg, depending on cultivar), tapioca starch (2.5 nmol per mg of starch), rice starch (1.0 nmol per mg of starch), mung bean starch (3.5 nmol per mg of starch) and sorghum starch (0.9 nmol per mg of starch). In barley starch and starch from various waxy mutants of corn, these authors were not able to detect any starch phosphate bonded in the C6 position. Up to now, it has not been possible to make any connection between the genotype of a plant and the content of starch phosphate (Jane et al., 1996, Cereal Foods World 41 (11), 827-832). Therefore it is not possible at present to influence the content of starch phosphate in plants by breeding measures.
Up to now, two proteins have been described which mediate the introduction of covalent bonds of phosphate radicals into the glucose molecules of starch. The first protein has the enzymatic activity of an alpha-glucan-water dikinase (GWD, E.C.: 2.7.9.4) (Ritte et al., 2002, PNAS 99, 7166-7171), is often called R1, in particular in the older scientific literature, and is bonded to the starch granules of the reserve starch in potato tubers (Lorberth et al., 1998, Nature Biotechnology 16, 473-477). The second protein described in the literature, which catalyzes the introduction of starch phosphate into starch, has the enzymatic activity of a phosphoglucan-water dikinase (PWD, E.C.: 2.7.9.5) (Kötting et al., 2005, Plant Physiol. 137, 2424-252, Baunsgaard et al., 2005, Plant Journal 41, 595-605).
A significant difference between GWD and PWD consists in the fact that GWD can use unphosphorylated starch as a substrate, i.e. a de novo phosphorylation of unphosphorylated starch can be catalyzed by GWD, whereas PWD needs already phosphorylated starch as a substrate, i.e. additionally introduces phosphate into already phosphorylated starch (Kötting et al., 2005, Plant Physiol. 137, 2424-252, Baunsgaard et al., 2005, Plant Journal 41, 595-605). A further significant difference between GWD and PWD consists in the fact that GWD introduces phosphate groups exclusively into the C6 position of the glucose molecules of starch, whereas PWD exclusively phosphorylates the C3 position of the glucose molecules of phosphorylated starch (Ritte et al., 2006, FEBS Letters 580, 4872-4876).
In the reaction catalyzed by GWD or PWD, the starting materials alpha-1,4-glucan (for GWD) or phosphorylated alpha-1,4-glucan (for PWD), adenosine triphosphate (ATP) and water are reacted to give the products glucan phosphate (starch phosphate), monophosphate and adenosine monophosphate (Kötting et al., 2005, Plant Physiol. 137, 2424-252, Ritte et al., 2002, PNAS 99, 7166-7171).
Wheat plants which have an increased activity of GWD proteins due to expression of a GWD-encoding gene from potato are described in WO 02 34923. In comparison to corresponding wild-type plants in which it was not possible to detect any starch phosphate, these plants synthesize a starch containing significant amounts of starch phosphate in the C6 position of the glucose molecules.
WO 05 2359 describes the overexpression of a GWD from potato in corn plants, optimized with respect to codons used by corn plants. By means of 31P NMR, a total phosphate content (bonded in the C6, C3 and C2 position of the glucose molecules) of the corn starch in question of 0.0736% phosphate based on the amount of glucose was determined. If a molecular weight of 98 is taken as a basis for phosphate, a total phosphate content of about 7.5 nmol of phosphate per mg of starch results for the total phosphate content determined in WO 05 2359 of 0.0736% for starch isolated from transgenic corn plants.
Plants which have an increased activity of a PWD protein due to overexpression of a PWD-encoding gene from Arabidopsis thaliana are described in WO 05 095617. In comparison to corresponding untransformed wild-type plants, these plants have an increased content of starch phosphate.
An important functional property, for example in the processing of starches in the food industry, is the swelling power. Various structural properties of starches, such as the amylose/amylopectin ratio, the side chain length, the molecular weight, the number of branchings, have an influence on the swelling power of the starches in question (Narayana and Moorthy, 2002, Starch/Stärke 54, 559-592).
The advice can be taken from the scientific literature that, in addition to the amylose/amylopectin ratio, the side chain distribution of the amylopectin and the molecular weight distribution of the starch polymers, also the amount of starch phosphate, has an influence on functional properties, in particular on the swelling power of the starch (Narayana and Moorthy, 2002, Starch/Stärke 54, 559-592).
It is to be emphasized that concerning the swelling power of starch a distinction is to be made between the swelling power in cold water (e.g. room temperature) and the swelling power in warm or hot water. Native starches have a negligible swelling power, if at all, in cold water, whereas physically modified (pregelatinized, dried) starches are already able to swell in cold water. Production processes for starches swelling in cold water are described, for example, in U.S. Pat. No. 4,280,851. In connection with the present invention, the term “swelling power” relates to the behavior of starch in warm/hot aqueous suspensions. The swelling power is standardly determined by warming starch granules in the presence of an excess of water, removing unbound water after centrifugation of the suspension and forming the quotient of the weight of the residue obtained and the weight of the amount of starch weighed in. When carrying out this process, on warming the starch suspension crystalline areas of the starch granules are dissolved and water molecules are intercalated in the starch granules, but without the structure of the starch granules itself being destroyed here, i.e. only a swelling of the individual starch granules, caused by the absorption of water molecules, takes place.
In comparison to cereal starches, starches isolated from tubers or tuberous tissues have a significantly higher hot water swelling power.
For potato starches isolated from various varieties, a maximum swelling power of 74.15 g/g (Kufri Jyoti variety) was determined at 85° C. (Singh et al., 2002, Journal of the Science of Food and Agriculture 82, 1376-1383) according to the method of Leach et al. (1959, Cereal Chemistry 36, 534-544). Takizawa et al. (2004, Brazilian Archives of Biology and Technology 47 (6), 921-931) determined a swelling power of 100 g/g for potato starch (90° C., according to the method of Leach et al. (1959, Cereal Chemistry 36, 534-544)). Wheat starch, isolated from various cultivars, has a swelling power of 16.6 g/g to 26.0 g/g (temperature: boiling aqueous 0.1% AgNO3 suspension) (Yamamori and Quynh, 2000, Theor Appl Genet 100, 23-28). Starch isolated from various cultivars of hull-less barley has a swelling power of 16.5 g/g or 19.3 g/g and waxy or amylose-free starch of the various cultivars of said barley has a swelling power of 36.0 g/g to 55.7 g/g (temperature: 70° C. aqueous 0.1% AgNO3, Yasui et al., 2002, Starch/Stärke 54, 179-184). For corn starch, a swelling power of 22.3 g/g and for high amylose corn starches a swelling power of 9.6 g/g (Hylon V), 6.1 g/g (Hylon VII) or 3.9 g/g (LAPS=Low AmyloPectin Starch) was determined (90° C., Shi et al., 1998, J. Cereal Sci. 27, 289-299). In U.S. Pat. No. 6,290 9,907, a swelling power of 35.4 g/g was indicated for waxy corn starch. For starch isolated from various rice cultivars, a swelling power of 26.0 g/g to 33.2 g/g was determined (Sodhi and Singh, 2003, Food Chemistry 80, 99-108) according to the method of Leach et al. (1959, Cereal Chemistry 36, 534-544). Chen et al. (2003, Starch/Stärke 55, 203-212) determined a swelling power of approximately 25 g/g to approximately 49 g/g (95° C., aqueous suspension) for various mixtures of waxy rice starches with high-amylose rice starches. Yasui et al. (2002, Starch/Stärke 54, 179-184) determined a swelling power of 55.7 g/g (measured in boiling water in 0.1% aqueous silver nitrate solution) for an amylose-free rice starch.
By the preparation of derivatives of native starches, functional properties of the starches can be altered. “Cross-linked” wheat starches, depending on the degree of cross-linking, have a swelling power of 6.8 g/g to 8.9 g/g, acetylated wheat starches have a swelling power of at most 10.3 g/g and at the same time cross-linked and acetylated wheat starches have a swelling power of 9.4 g/g, whereas the corresponding underivatized starches had a swelling power of 8.8 g/g (measured at 90° C.; Van Hung and Morita, 2005, Starch/Stärke 57, 413-420).
For acetylated waxy rice starches, a swelling power of about 30 g/g and for cross-linked waxy rice starch a swelling power of about 15 g/g was determined, whereas corresponding underivatized waxy rice starch had a swelling power of about 41 g/g. Acetylated rice starch had a swelling power of about 20 g/g and cross-linked rice starch had a swelling power of about 13 g/g, whereas corresponding underivatized rice starch had a swelling power of about 14 g/g (measured at 90° C., Liu et al., 1999, Starch/Stärke 52, 249-252). U.S. Pat. No. 6,299,907 describes cross-linked starches, the cross-linking reaction being carried out after pre-swelling of the starches in question in a sodium hydroxide/sulfate solution. Depending on the degree of crosslinkage, for wheat starch a swelling power of 6.8 g/g to 7.3 g/g (corresponding underivatized wheat starch 14.7 g/g), for hydroxypropyl-wheat starch a swelling power of 9.7 g/g (corresponding underivatized wheat starch 22.9 g/g), for cross-linked corn starch a swelling power of 5.9 g/g (corresponding underivatized corn starch 16.7 g/g), for cross-linked waxy corn starch a swelling power of 8.3 g/g (corresponding underivatized waxy corn starch 35.4 g/g) and for cross-linked potato starch a swelling power of 6.7 g/g (corresponding underivatized potato starch was not accurately specified) was determined (measured at 95° C.). It results from this that the swelling power of starch cannot be increased significantly, if at all, by methods of derivatization customary nowadays.